1. Field of the Invention
This invention relates to methods for developing and utilizing antibodies, and more specifically to methods for developing and using antibodies with preferential recognition of phenylboronic acid complexes of glycosylated proteins.
2. Background of the Invention
Stasis of circulating plasma glucose levels is principally attributed to hormonal regulation. In the normal individual, this balance is achieved through the regulation of the antagonistic activity of insulin and glucagon. The diabetic patient has diminished or absent production of insulin which results in excess levels of glucose within the circulation. Treatments with exogenous insulin therapy are intended to reinstate control against excessive plasma glucose levels. Diagnostic methods for monitoring the long-term stability of blood glucose levels are important in the clinical treatment and maintenance of diabetic patients.
A chemical property of glucose is that the molecule can exist as a pyranose ring or linearized aldehyde. The aldehyde form can form a Schiff base structure when combined with 1.degree. amines such as those found on the amino acid lysine or amino acid residues at the N-terminal end of proteins. This intermediate structure may reversibly dissociate to free glucose and the protein, or a chemical rearrangement (known as an Amadori rearrangement) can take place resulting in a new stabile chemical structure.
The level of glycation of proteins represents the average of the rises and declines in concentration of blood glucose and the length of time in contact with said proteins. Many different proteins can react with glucose as described leading to a subpopulation of proteins defined by the level of glycation. Several proteins have proven to be useful as markers for monitoring this glycation reaction. The protein hemoglobin has been a particularly useful and well acted marker for monitoring the efficacy of long-term insulin therapy. This is because the long half-life of hemoglobin (which can be up to 120 days) permits the assessment of average blood glucose levels over extended periods of time. Normal patients have a glycated hemoglobin content of 4-7% of the total, whereas untreated diabetics have levels of 12-20%. Clinicians use glycated hemoglobin levels to monitor the maintenance of normal physiologic glucose levels over extended time intervals, and as a predictive aid for other advanced sequelae of a prolonged hyperglycemic condition.
Hemoglobin exists as a tetramer comprised of two a and two b subunits. The molecule can be glycated at the N-terminal valine residues resident in either of the subunits. Additionally, the molecule can be glycated at any of several lysine moieties resident with either subunit. The level of glycation at each amino acid residue, while relatively proportional, is not equivalent.
Glycated hemoglobin has been measured in several ways including electrophoresis, cation exchange chromatography, affinity chromatography and by immunoassay. Each method has its benefits and detracting points. Electrophoresis can allow ready separation of the modified protein by relative mobility in an electric field or to its isoelectric point, however, variant forms of hemoglobin (for example, HbS) can result in spurious migration relative to normal forms of the molecule. Cation exchange chromatography can also afford ready separation of the protein but is very sensitive to ionic strength, pH and other sample loading conditions, and is again affected by aberrant hemoglobin variants. Affinity chromatography by capture and elution of the glycated fraction on a phenylboronate column is not affected by variant forms of the protein and is as such the most widely accepted method. However, affinity chromatography as with electrophoretic and chromatographic procedures are relatively laborious, difficult to standardize and cumbersome to automate.
Immunoassay methods have received considerable interest because they are particularly amenable to automation. Several strategies for development of such assays have been described and are well known to those practiced in the science of diagnostic immunochemistry. One such example depends on the use of an antibody directed toward the glycated form of the N-terminal valine on the hemoglobin , .beta.-chain. This determinant, designated HbA.sub.1c, is the most prevalent glycation site on hemoglobin representing approximately 40-45% of the total glycated content of the molecule. Several representative patents cover the immunologic recognition of this determinant including U.S. Pat. No. 4,647,654, U.S. Pat. No. 4,727,036, U.S. Pat. No. 4,658,022, U.S. Pat. No. 4,247,533, U.S. Pat. No. 4,478,744, U.S. Pat. No. 4,970,171, U.S. Pat. No. 5,206,144 and EP 329994.
The method described in U.S. Pat. No. 4,647,654 relates to the preparation of a glycated immunogenic synthetic antigen by contacting a peptide fragment representing the N-terminal amino acid sequence of HbA.sub.1c with glucose over an extending time interval, isolating the glycated fraction, coupling the fraction onto a carrier protein for presentation to an animal host. U.S. Pat. No. 4,727,036 describes a method for using monoclonal antibodies raised with the aforementioned antigen in developing an immunoassay to measure HbA.sub.1c. Documents U.S. Pat. No. 4,970,171 and U.S. Pat. No. 4,658,022 describe denaturants and methods of denaturing hemoglobin so as to expose the HbA.sub.1c epitope for recognition by the aforementioned antibody. U.S. Pat. No. 4,247,533 describes a radioimmunoassay for detection of HbA.sub.1c. U.S. Pat. No. 4,478,744 describes a method for raising polyclonal antibodies to HbA.sub.1c. U.S. Pat. No. 5,206,144 describes a method for determining HbA.sub.1c levels without the need for chemical denaturation. And EP 329994 describes a synthetic antigen for raising antibodies to HbA.sub.1c that depends on the use of very specific chemical linker strategies.
While the high level of specificity of antibodies to HbA.sub.1c is a strength in methods dependent upon the same, said specificity has also proven to be a weakness as well. In particular, most such antibodies described to date have been found to show bias toward the recognition of normal forms of HbA.sub.1c and to a lesser extent or not at all with the variant forms of the protein. This behavior would be undesirable since it would result in the underestimation of blood HbA.sub.1c levels.
Accordingly there is a need to develop a reagent which draws from the recognized advantages of the phenylboronate chemistry while providing the advantages of an immunochemical approach.